Rhodospirillum rubrum responds to the presence of carbon monoxide (CO) in its environment by expressing a set of genes whose products oxidize CO to CO2 and generate energy in the process. CooA is the protein that is necessary for this CO-dependent transcription, and is a homolog of CRP. In the previous grant period, we have purified CooA to homogeneity and shown it to be a dimer of approximately 54 kD that contains protoheme as the CO- binding site. Because CooA is a dimer under all conditions, but only binds DNA upon CO binding, it is likely that CooA undergoes a conformational change analogous to that of CRP upon cAMP binding. The heme of CooA is 6-coordinate before and after CO binding, suggesting that ligand displacement by CO is the likely trigger for that conformational change. CooA must be reduced in order to bind CO and an unusual ligand switch occurs upon reduction. In the present proposal, we will determine the molecular basis for the response of CooA to CO by a variety of mutational, spectroscopic and structural analyses. In the course of this, we will identify the remaining unknown ligand to the heme and the other residues critical for the response to CO. These include the residues that are involved in the redox-dependent ligand switch, as well as those involved in the conformational change itself. Among the many interesting properties of CooA is its specificity for CO and its failure to respond functionally to other small molecules that typically bind hemes. We propose a variety of approaches to understand this specificity including a mutagenesis approach which is highly likely to identify CooA variants of novel properties. Subsequent biochemical and spectral analysis of these variants should not only reveal the important features of CooA activity, but suggest general rules for the binding of small molecules to hemes. The ability to select novel variants of CooA through the use of reporter systems is an enormous technical advantage compared to the case with many other heme proteins. This self-reporting property will be exploited to gain an insight into the molecular basis for heme reactivity and function in heme proteins of medical interest, such as hemoglobins and soluble guanylyl cyclase.